Immobilized microbial nitrilase for production of glycolic acid

ABSTRACT

The present invention provides a process for preparing an enzyme catalyst having nitrilase activity for hydrolysis of glycolonitrile to glycolic acid with improved retention of recovered catalyst activity in consecutive batch reactions with catalyst recycle, said process comprising pretreating the enzyme catalyst with glutaraldehyde. The glutaraldehyde-pretreated enzyme catalyst has improved specific activity when compared to non-glutaraldehyde-pretreated enzyme catalysts, and thereby, has improved overall catalyst activity and productivity.

FIELD OF THE INVENTION

This invention relates to the field of organic acid synthesis andmicrobiology. More specifically, the present invention provides aprocess for preparing an enzyme catalyst having improved retention ofinitial nitrilase activity during hydrolysis of glycolonitrile toglycolic acid, said process comprising pretreating the enzyme catalystwith glutaraldehyde prior to immobilization and cross-linking. Theglutaraldehyde-pretreated immobilized and cross-linked enzyme catalysthas improved specific activity, as stated above, when compared tonon-glutaraldehyde-pretreated immobilized and cross-linked enzymecatalysts, and thereby, has improved overall catalyst activity, catalystproductivity and volumetric productivity for the conversion ofglycolonitrile to glycolic acid.

BACKGROUND OF THE INVENTION

Glycolic acid (HOCH₂COOH; CAS Registry Number is 79-14-1) is thesimplest member of the α-hydroxy acid family of carboxylic acids. Itsproperties make it ideal for a broad spectrum of consumer and industrialapplications, including use in water well rehabilitation, the leatherindustry, the oil and gas industry, the laundry and textile industry, asa monomer in the preparation of polyglycolic acid (PGA), and as acomponent in personal care products. Glycolic acid also is a principleingredient for cleaners in a variety of industries (dairy and foodprocessing equipment cleaners, household and institutional cleaners,industrial cleaners [for transportation equipment, masonry, printedcircuit boards, stainless steel boiler and process equipment, coolingtower/heat exchangers], and metals processing [for metal pickling,copper brightening, etching, electroplating, electropolishing]). It hasalso been reported that polyglycolic acid is useful as a gas barriermaterial (i.e., exhibits high oxygen barrier characteristics) forpacking foods and carbonated drinks (WO 2005/106005 A1). However,traditional chemical synthesis of glycolic acid produces a significantamount of impurities that must be removed prior to use. New technologyto commercially produce glycolic acid, especially one that producesglycolic acid in high purity and at low cost, would be eagerly receivedby industry.

Microbial enzyme catalysts can hydrolyze a nitrile (e.g.,glycolonitrile) directly to the corresponding carboxylic acids (e.g.,glycolic acid) using a nitrilase (EC 3.5.5.7), where there is nointermediate production of the corresponding amide (Equation 1), or by acombination of nitrile hydratase (EC 4.2.1.84) and amidase (EC 3.5.1.4)enzymes, where a nitrile hydratase (NHase) initially converts a nitrileto an amide, and then the amide is subsequently converted by the amidaseto the corresponding carboxylic acid (Equation 2):

It has been demonstrated that the enzyme catalyst specific activitymeasured as micromoles glycolic acid produced per minute per g dry cellweight of catalyst, decreases by from 35% to 50% of initial activityafter a single use in consecutive batch reactions with biocatalystrecycle. This represents a significant loss in specific activity of theenzyme catalyst in batch reactions, and, in turn, overall enzymeactivity and productivity. For a commercially feasible enzymatic processfor producing glycolic acid, this loss in enzyme activity needs to beaddressed.

One aspect of the loss of enzyme activity may be attributable toimpurities and other components present when reacting the enzymecatalyst with glycolonitrile for glycolic acid production. Methods tosynthesize glycolonitrile by reacting aqueous solutions of formaldehydeand hydrogen cyanide have previously been reported (U.S. Pat. No.2,175,805; U.S. Pat. No. 2,890,238; and U.S. Pat. No. 5,187,301;Equation 3).

However, these methods typically result in an aqueous glycolonitrilereaction product that requires significant purification (e.g.,distillative purification) as many of the impurities and/or byproductsof the reaction (including excess reactive formaldehyde) may interferewith the enzymatic conversion of glycolonitrile to glycolic acid,including suppression of catalyst activity (i.e., decreased specificactivity). In particular, it is well known that formaldehyde can createundesirable modifications in proteins by reacting with amino groups fromN-terminal amino acid residues and the side chains of arginine,cysteine, histidine, and lysine residues (Metz et al., J. Biol. Chem.,279 (8): 6235-6243 (2004)). Suppression of catalyst activity decreasesthe overall productivity of the catalyst (i.e., total grams of glycolicacid formed per gram of catalyst), adding a significant cost to theoverall process that may make enzymatic production economicallynon-viable when compared to chemical synthesis. As such, reactionconditions are needed that can help to protect the enzymatic activityagainst undesirable impurities that decrease the activity of thecatalyst.

A method of producing high purity glycolonitrile has been reported bysubjecting the formaldehyde to a heat treatment prior to theglycolonitrile synthesis reaction (U.S. application Ser. No. 11/3143865and U.S. application Ser. No. 11/314905; Equation 3). However,glycolonitrile can reversibly disassociate into formaldehyde andhydrogen cyanide. As such, there remains a need to protect nitrilaseactivity against the undesirable effects of both formaldehyde andhydrogen cyanide produced by dissociation of glycolonitrile.

U.S. Pat. No. 5,508,181 also describes similar difficulties related torapid enzyme catalyst inactivation when converting nitrile compounds toα-hydroxy acids. Specifically, U.S. Pat. No. 5,508,181 provides thatα-hydroxy nitrile compounds partially disassociate into thecorresponding aldehydes, according to the disassociation equilibrium.These aldehydes were reported to inactivate the enzyme within a shortperiod of time by binding to the protein, thus making it difficult toobtain α-hydroxy acid or α-hydroxy amide in a high concentration withhigh productivity from α-hydroxy nitriles (col. 2, lines 16-29). As asolution to prevent enzyme inactivation due to accumulation ofaldehydes, phosphate or hypophosphite ions were added to the reactionmixture. Similarly, U.S. Pat. No. 5,326,702 describes the use ofsulfite, disulfite, or dithionite ions to sequester aldehyde and preventenzyme inactivation, but concludes that the concentration of α-hydroxyacid produced and accumulated even by using such additives is notsufficient for most commercial purposes.

Moreover, U.S. Pat. No. 6,037,155 teaches that low accumulation ofα-hydroxy acid product is related to enzyme inactivation within a shorttime due to the disassociated-aldehyde accumulation. These inventorssuggest that enzymatic activity is inhibited in the presence of hydrogencyanide (Asano et al., Agricultural Biological Chemistry, Vol. 46, pages1165-1174 (1982)) generated in the partial disassociation of theα-hydroxy nitrile in water together with the corresponding aldehyde orketone (Mowry, David T., Chemical Reviews, Vol. 42, pages 189-283(1948)). The inventors address the problem of aldehyde-induced enzymeinactivation by using microorganisms whose enzyme activity could beimproved by adding a cyanide substance to the reaction mixture. Theaddition of a cyanide substance limited the disassociation of α-hydroxynitrile to aldehyde and hydrogen cyanide. While this tactic provides abenefit to the system, it only addresses one aspect associated withenzyme inactivation in conversion of glycolonitrile to glycolic acid, inthat, as stated above, glycolonitrile is known to reversiblydisassociate to hydrogen cyanide and formaldehyde, and both are known tonegatively effect enzyme catalyst activity.

A separate process has been developed to protect the specific activityof an enzyme catalyst having nitrilase activity when convertingglycolonitrile to glycolic acid in the presence of formaldehyde (seecopending U.S. application Ser. No. 11/931,069 incorporated herein byreference), where significant improvements in catalyst activity andstability were achieved by adding an amine protectant to the reactionmixture, or by immobilization of the nitrilase catalyst in or on amatrix that is comprised of an amine protectant, e.g. PEI,polyallylamine, PVOH/polyvinylamine, etc. In that system, the specificactivity of the catalyst in the presence of formaldehyde is improved,but does not address, altogether, issues related to the loss in specificactivity of recovered catalyst activity in consecutive batch reactionswith catalyst recycle.

U.S. Pat. No. 4,288,552 discloses (column 1, lines 46-49, and column 2,lines 50-55) that glutaraldehyde-sensitive enzymes (such asthiol-enzymes (e.g, nitrilase) and others with an SH group in or verynear the active site of the enzyme molecule) are inactivated bythiol-reactive agents such as glutaraldehyde. Therefore, use ofglutaraldehyde to improve the retention of initial catalyst activityduring hydrolysis of glycolonitrile to glycolic acid was heretoforeunpredictable. Said unpredictable benefit is demonstrated herein.

Therefore there is a need for a process that provides improved retentionof initial nitrilase activity during hydrolysis of glycolonitrile toglycolic acid, thereby improving overall catalyst activity, catalystproductivity, and volumetric productivity for the conversion ofglycolonitrile to glycolic acid.

SUMMARY OF THE INVENTION

The present invention resolves the need described above by providing aprocess for preparing an enzyme catalyst having nitrilase activity withimproved retention of initial catalyst activity for hydrolysis ofglycolonitrile to glycolic acid, said process comprising pretreating theenzyme catalyst with glutaraldehyde prior to immobilization andcross-linking. The glutaraldehyde-pretreated immobilized andcross-linked enzyme catalyst has improved specific activity during theconversion of glycolonitrile to glycolic acid when compared toimmobilized and cross-linked enzyme catalysts prepared withoutglutaraldehyde-pretreatment, and thereby, has improved overall catalystactivity, catalyst productivity and volumetric productivity for theconversion of glycolonitrile to glycolic acid.

In one aspect, the present invention provides a process for producing anenzyme catalyst having nitrilase activity with improved retention of theinitial specific activity during the conversion of glycolonitrile toglycolic acid, said process comprising:

-   -   (a) producing an enzyme catalyst having nitrilase activity by        fermentation;    -   (b) pretreating said enzyme catalyst with glutaraldehyde;    -   (c) optionally inactivating unreacted glutaraldehyde with        bisulfite following glutaraldehyde pretreatment;    -   (d) recovering the enzyme catalyst from (b) or (c), and        immobilizing said enzyme catalyst in carrageenan; and    -   (e) cross-linking the resulting carrageenan-immobilized enzyme        catalyst of (d) with glutaraldehyde and polyethylenimine,        whereby a glutaraldehyde-pretreated immobilized and cross-linked        enzyme catalyst is produced and wherein said        glutaraldehyde-pretreated immobilized and cross-linked enzyme        catalyst has improved retention of initial specific activity        during conversion of glycolonitrile to glycolic acid as compared        to non-glutaraldehyde-pretreated immobilized and cross-linked        enzyme catalysts under the same reaction conditions.

A further aspect of the invention comprises contacting theglutaraldehyde-pretreated immobilized and cross-linked enzyme catalystof (e) above with glycolontrile in an aqueous solution, under suitablereaction conditions whereby glycolic acid is produced. And further,recovering said glycolic acid.

Another aspect of the invention is directed to theglutaraldehyde-pretreated immobilized and cross-linked enzyme catalystthat is produced by the process of steps a) through e) above. Further,said glutaraldehyde-pretreated immobilized and cross-linked enzymecatalyst, having nitrilase activity, retains a significantly-greaterpercentage of its initial specific activity (μmoles of glycolonitrilehydrolyzed per minute per gram of catalyst) when used for the conversionof glycolonitrile to glycolic acid (as the ammonium salt) as compared toa comparable enzyme catalyst without glutaraldehyde pretreatment.

Another aspect of the invention is directed to aglutaraldehyde-pretreated immobilized and cross-linked enzyme catalysthaving nitrilase activity that is produced by the process of steps a)through e) above, wherein said enzyme catalyst retains at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 96%, or at leastabout 97% of its initial specific activity after at least fourconsecutive batch recycles.

Another aspect of the invention is directed to aglutaraldehyde-pretreated immobilized and cross-linked enzyme catalysthaving nitrilase activity that is produced by the process of steps a)through e) above, wherein said enzyme catalyst retains at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 96%, or at leastabout 97% of its initial specific activity after the production of atleast 40 g of glycolic acid per gram dry cell weight ofglutaraldehyde-pretreated immobilized and cross-linked enzyme catalystin a continuous reaction for the production of glycolic acid fromglycolonitrile, for example, when running the reaction in a continuouslystirred tank reactor (CSTR), or in a fixed-bed plug flow reactor, or ina fluidized-bed or semi-fluidized bed reactor.

A further aspect of the invention is directed to an improved process forhydrolyzing glycolonitrile to glycolic acid comprising improving theretention of the initial specific activity of an immobilized enzymecatalyst having nitrilase activity during conversion of glycolonitrileto glycolic acid, said process comprising:

-   -   (a) producing an enzyme catalyst having nitrilase activity by        fermentation;    -   (b) pretreating said enzyme catalyst with glutaraldehyde;    -   (c) optionally inactivating unreacted glutaraldehyde with        bisulfite following glutaraldehyde pretreatment;    -   (d) recovering the enzyme catalyst from (b) or (c) and        immobilizing said enzyme catalyst in carrageenan;    -   (e) cross-linking the resulting carrageenan-immobilized enzyme        catalyst of (d) with glutaraldehyde and polyethylenimine,        whereby a cross-linked immobilized enzyme catalyst is produced;        and    -   (f) contacting the cross-linked immobilized enzyme catalyst of    -   (e) with glycolontrile in an aqueous solution under suitable        reaction conditions whereby glycolic acid is produced,        wherein step (f) occurs in at least two consecutive batch        recycles.

Further, the process may include recovering the glycolic acid producedby said improved process.

BRIEF DESCRIPTION OF THE FIGURE, SEQUENCE LISTING, AND THE BIOLOGICALDEPOSITS

The invention can be more fully understood from the Figure, sequencelisting, biological deposits, and detailed description, that togetherform this application.

FIGURE

FIG. 1, panels A-G, is a CLUSTALW alignment (version 1.83 using defaultparameters) of various nitrilase sequences. The conserved catalystsignature sequence surrounding the catalyst cysteine residue ishighlighted in gray shading. The amino acids representing the catalytictriad (Glu₄₈, Lys₁₃₀, and Cys₁₆₄; numbering based on the amino acidsequence SEQ ID NO: 4) are underlined.

SEQUENCE LISTING

The following sequence descriptions and sequences listings attachedhereto comply with the rules governing nucleotide and/or amino acidsequence disclosures in patent applications as set forth in 37 C.F.R.§1.821-1.825. The Sequence Descriptions contain the one letter code fornucleotide sequence characters and the three letter codes for aminoacids as defined in conformity with the IUPAC-IYUB standards describedin Nucleic Acids Research 13:3021-3030 (1985) and in the BiochemicalJournal 219 (No. 2):345-373 (1984) which are herein incorporated byreference. The symbols and format used for nucleotide and amino acidsequence data comply with the rules set forth in 37 C.F.R. §1.822.

SEQ ID NO: 1 is the amino acid sequence of the catalytic signature motifencompassing the essential cysteine residue of nitrilase enzymes(Formula 1).

SEQ ID NO: 2 is the amino acid sequence of a preferred catalystsignature motif encompassing the essential cysteine residue of nitrilaseenzymes (Formula 2).

SEQ ID NO: 3 is the nucleotide sequence of the Acidovorax facilis 72Wnitrilase coding sequence comprising a change in the start codon fromTTG to ATG to facilitate recombinant expression in E. coli.

SEQ ID NO: 4 is the deduced amino acid sequence of the Acidovoraxfacilis 72W nitrilase (ATCC 55746).

SEQ ID NO: 5 is the amino acid sequence of the Alcaligenes faecalis JM3nitrilase (GENBANK® BAA02684.1).

SEQ ID NO: 6 is the amino acid sequence of the Rhodococcus rhodochrousJ1 nitrilase (GENBANK® Q03217).

SEQ ID NO: 7 is the amino acid sequence of the Rhodococcus rhodochrousK22 nitrilase (GENBANK® Q02068).

SEQ ID NO: 8 is the amino acid sequence of the Nocardia sp. C-14-1nitrilase (GENBANK® AAX18182.1).

SEQ ID NO: 9 is the amino acid sequence of the Bordetella bronchisepticaRB50 nitrilase (GENBANK® NP_(—)887662.1).

SEQ ID NO: 10 is the amino acid sequence of the Arabidopsis thaliananitrilase (GENBANK AAB60275.1 and AAA19627.1).

SEQ ID NO: 11 is the amino acid sequence of the Synechococcus elongatusPCC 7942 nitrilase (GENBANK® YP_(—)399857.1).

SEQ ID NO: 12 is the amino acid sequence of the Synechococcus elongatusPCC 6301 nitrilase (GENBANK®YP_(—)171411.1).

SEQ ID NO: 13 is the amino acid sequence of the Synechocystis sp. PCC6803 nitrilase (GENBANK® NP_(—)442646.1).

SEQ ID NO: 14 is the amino acid sequence of the Pseudomonas entomophilaL48 nitrilase (GENBANK® YP_(—)6090481.1).

SEQ ID NO: 15 is the amino acid sequence of the Zymomonas moblisnitrilase (GENBANK® YP_(—)162942.1).

SEQ ID NO: 16 is the amino acid sequence of the Bacillus sp. 0xB-1nitrilase (GENBANK® BAA90460.1).

SEQ ID NO: 17 is the amino acid sequence of the Comamonas testosteroninitrilase (GENBANK® AAA82085.1).

SEQ ID NO: 18 is the amino acid sequence of the Synechococcus sp. CC9605nitrilase (GENBANK® YP⁻381420.1).

SEQ ID NO: 19 is the amino acid sequence of the Pseudomonas fluorescensPf-5 nitrilase (GENBANK® YP_(—)260015.1).

SEQ ID NO: 20 is the amino acid sequence of the Nocardia farcinica IFM10152 nitrilase (GENBANK® YP_(—)119480.1).

SEQ ID NO: 21 is the amino acid sequence of the Alcaligenes faecalis1650 nitrilase (GENBANK® AAY06506.1).

SEQ ID NO: 22 is the amino acid sequence of the Pseudomonas syringae pv.syringae B728a nitrilase (GENBANK® AAY35081.1).

SEQ ID NO: 23 is the amino acid sequence of the Bradyrhizobium sp. BTAilnitrilase (GENBANK® ZP_(—)00859948.1).

SEQ ID NO: 24 is the amino acid sequence of the Rhodococcus rhodochrousNCIMB 11216 nitrilase (GENBANK® CAC88237).

SEQ ID NO: 25 is the amino acid sequence of Rhodococcus rhodochrousATCC™ 39484

SEQ ID NO: 26 is the nucleotide sequence of an A. facilis 72W nitrilasemutant comprising a codon change which resulted in a single amino acidsubstitution at residue position 201 (L201Q; Leu

 Gln).

SEQ ID NO: 27 is the deduced amino acid sequence of the mutant nitrilase(SEQ ID NO: 26) comprising a single amino acid substitution at residueposition 201 (Leu201

 Gln) of the A. facilis 72W nitrilase.

SEQ ID NO: 28 is the nucleotide sequence of an A. facilis 72W nitrilasemutant comprising a codon change which resulted in a single amino acidsubstitution at residue position 201 (L201A; Leu

 Ala).

SEQ ID NO: 29 is the deduced amino acid sequence of the mutant nitrilase(SEQ ID NO: 28) comprising a single amino acid substitution at residueposition 201 (Leu201

 Ala) of the A. facilis 72W nitrilase.

SEQ ID NO: 30 is the nucleotide sequence of an A. facilis 72W nitrilasemutant comprising a codon change which resulted in a single amino acidsubstitution at residue position 201 (L201C; Leu

 Cys).

SEQ ID NO: 31 is the deduced amino acid sequence of the mutant nitrilase(SEQ ID NO: 30) comprising a single amino acid substitution at residueposition 201 (Leu201

 Cys) of the A. facilis 72W nitrilase.

SEQ ID NO: 32 is the nucleotide sequence of an A. facilis 72W nitrilasemutant comprising a codon change which resulted in a single amino acidsubstitution at residue position 201 (L201T; Leu

 Thr).

SEQ ID NO: 33 is the deduced amino acid sequence of the mutant nitrilase(SEQ ID NO: 32) comprising a single amino acid substitution at residueposition 201 (Leu201

 Thr) of the A. facilis 72W nitrilase.

SEQ ID NO: 34 is the nucleotide sequence of an A. facilis 72W nitrilasemutant comprising a codon change which resulted in a single amino acidsubstitution at residue position 201 (L201G; Leu

 Gly).

SEQ ID NO: 35 is the deduced amino acid sequence of the mutant nitrilase(SEQ ID NO: 34) comprising a single amino acid substitution at residueposition 201 (Leu201

 Gly) of the A. facilis 72W nitrilase.

SEQ ID NO: 36 is the nucleotide sequence of an A. facilis 72W nitrilasemutant comprising a codon change which resulted in a single amino acidsubstitution at residue position 201 (L201H; Leu

 His).

SEQ ID NO: 37 is the deduced amino acid sequence of the mutant nitrilase(SEQ ID NO: 36) comprising a single amino acid substitution at residueposition 201 (Leu201

 His) of the A. facilis 72W nitrilase.

SEQ ID NO: 38 is the nucleotide sequence of an A. facilis 72W nitrilasemutant comprising a codon change which resulted in a single amino acidsubstitution at residue position 201 (L201K; Leu

 Lys).

SEQ ID NO: 39 is the deduced amino acid sequence of the mutant nitrilase(SEQ ID NO: 38) comprising a single amino acid substitution at residueposition 201 (Leu201

 Lys) of the A. facilis 72W nitrilase.

SEQ ID NO: 40 is the nucleotide sequence of an A. facilis 72W nitrilasemutant comprising a codon change which resulted in a single amino acidsubstitution at residue position 201 (L201N; Leu

 Asn).

SEQ ID NO: 41 is the deduced amino acid sequence of the mutant nitrilase(SEQ ID NO: 40) comprising a single amino acid substitution at residueposition 201 (Leu201

 Asn) of the A. facilis 72W nitrilase.

SEQ ID NO: 42 is the nucleotide sequence of an A. facilis 72W nitrilasemutant comprising a codon change which resulted in a single amino acidsubstitution at residue position 201 (L201S; Leu

 Ser).

SEQ ID NO: 43 is the deduced amino acid sequence of the mutant nitrilase(SEQ ID NO: 42) comprising a single amino acid substitution at residueposition 201 (Leu201

 Ser) of the A. facilis 72W nitrilase.

SEQ ID NO: 44 is the nucleotide sequence of an A. facilis 72W nitrilasemutant comprising a codon change which resulted in a single amino acidsubstitution at residue position 168 (F168K; Phe

 Lys).

SEQ ID NO: 45 is the deduced amino acid sequence of the mutant nitrilase(SEQ ID NO: 44) comprising a single amino acid substitution at residueposition 168 (Phe168

 Lys) of the A. facilis 72W nitrilase.

SEQ ID NO: 46 is the nucleotide sequence of an A. facilis 72W nitrilasemutant comprising a codon change which resulted in a single amino acidsubstitution at residue position 168 (F168M; Phe

 Met).

SEQ ID NO: 47 is the deduced amino acid sequence of the mutant nitrilase(SEQ ID NO: 46) comprising a single amino acid substitution at residueposition 168 (Phe168

 Met) of the A. facilis 72W nitrilase.

SEQ ID NO: 48 is the nucleotide sequence of an A. facilis 72W nitrilasemutant comprising a codon change which resulted in a single amino acidsubstitution at residue position 168 (F168T; Phe

 Thr).

SEQ ID NO: 49 is the deduced amino acid sequence of the mutant nitrilase(SEQ ID NO: 48) comprising a single amino acid substitution at residueposition 168 (Phe168

 Thr) of the A. facilis 72W nitrilase.

SEQ ID NO: 50 is the nucleotide sequence of an A. facilis 72W nitrilasemutant comprising a codon change which resulted in a single amino acidsubstitution at residue position 168 (F168V; Phe

 Val).

SEQ ID NO: 51 is the deduced amino acid sequence of the mutant nitrilase(SEQ ID NO:50) comprising a single amino acid substitution at residueposition 168 (Phe168

 Val) of the A. facilis 72W nitrilase.

SEQ ID NO: 52 is the nucleotide sequence of an A. facilis 72W nitrilasemutant comprising a codon change which resulted in a single amino acidsubstitution at residue position 168 (T210A; Thr

 Ala).

SEQ ID NO: 53 is the deduced amino acid sequence of the mutant nitrilase(SEQ ID NO: 52) comprising a single amino acid substitution at residueposition 210 (Thr210

 Ala) of the A. facilis 72W nitrilase.

SEQ ID NO: 54 is the nucleotide sequence of an A. facilis 72W nitrilasemutant comprising a codon change which resulted in a single amino acidsubstitution at residue position 168 (T210C; Thr

 Cys).

SEQ ID NO: 55 is the deduced amino acid sequence of the mutant nitrilase(SEQ ID NO: 54) comprising a single amino acid substitution at residueposition 210 (Thr210

 Cys) of the A. facilis 72W nitrilase.

SEQ ID NO: 56 is the nucleotide sequence of the A. facilis 72W nitrilaseexpressed in E. coli strain SS1001 (ATCC PTA-1177).

SEQ ID NO: 57 is the deduced amino acid sequence of the mutant A.facilis 72W nitrilase expressed in E. coli SS1001 (ATCC PTA-1177).

BIOLOGICAL DEPOSITS

The following biological deposits have been made under the terms of theBudapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procedure:

Depositor Identification Int'l. Depository Reference Designation Date ofDeposit Acidovorax facilis 72W ATCC 55746 8 Mar. 1996 E. coli SS1001ATCC PTA-1177 11 Jan. 2000

As used herein, “ATCC” refers to the American Type Culture CollectionInternational Depository Authority located at ATCC, 10801 UniversityBlvd., Manassas, Va. 20110-2209, USA. The “International DepositoryDesignation” is the accession number to the culture on deposit withATCC.

The listed deposits will be maintained in the indicated internationaldepository for at least thirty (30) years and will be made available tothe public upon the grant of a patent disclosing it. The availability ofa deposit does not constitute a license to practice the subjectinvention in derogation of patent rights granted by government action.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a process for preparing an immobilizedand cross-linked enzyme catalyst having nitrilase activity forhydrolysis of glycolonitrile to glycolic acid with improved retention ofinitial enzyme catalyst activity during conversion of glycolonitrile toglycolic acid, said process comprising pretreating the enzyme catalystwith glutaraldehyde prior to immobilization. Theglutaraldehyde-pretreated immobilized enzyme catalyst has improvedretention of initial specific activity, as stated above, when comparedto the retention of initial specific activity ofnon-glutaraldehyde-pretreated immobilized and cross-linked enzymecatalysts during the conversion of glycolonitrile to glycolic acid, andthereby, has improved overall catalyst activity, catalyst productivityand volumetric productivity.

Definitions:

In this disclosure, a number of terms and abbreviations are used. Thefollowing definitions apply unless specifically stated otherwise.

As used herein, the term “comprising” means the presence of the statedfeatures, integers, steps, or components as referred to in the claims,but that it does not preclude the presence or addition of one or moreother features, integers, steps, components or groups thereof.

As used herein, the term “about” modifying the quantity of an ingredientor reactant of the invention employed refers to variation in thenumerical quantity that can occur, for example, through typicalmeasuring and liquid handling procedures used for making concentrates oruse solutions in the real world; through inadvertent error in theseprocedures; through differences in the manufacture, source, or purity ofthe ingredients employed to make the compositions or carry out themethods; and the like. The term “about” also encompasses amounts thatdiffer due to different equilibrium conditions for a compositionresulting from a particular initial mixture. Whether or not modified bythe term “about”, the claims include equivalents to the quantities. Inone embodiment, the term “about” means within 10% of the reportednumerical value, preferably within 5% of the reported numerical value.

As used herein, the term “glycolonitrile” is abbreviated as “GLN” and issynonymous with hydroxyacetonitrile, 2-hydroxyacetonitrile,hydroxymethylnitrile, and all other synonyms of CAS Registry Number107-16-4.

As used herein, the term “glycolic acid” is abbreviated as “GLA” and issynonymous with hydroxyacetic acid, hydroxyethanoic acid, and all othersynonyms of CAS Registry Number 79-14-1. The glycolic acid produced bythe present processes may in the form of the protonated carboxylic acidand/or the corresponding ammonium salt.

As used herein, the term “ammonium glycolate” is abbreviated “NH₄GLA”.

As used herein, the term “glycolamide” is the amide derived from thereaction of ammonia with glycolic acid and refers to all other synonymsof compounds having CAS Registry Number 598-42-5.

As used herein, the term “glycolide” refers to the compound of CASRegistry Number 502-97-6.

As used herein, the term “formaldehyde” is abbreviated as “FA” and issynonymous with formic aldehyde, methyl aldehyde, oxomethane, and allother synonyms of CAS Registry Number 50-00-0. Commercially availableformaldehyde is typically comprised of a mixture of monomericformaldehyde (“free formaldehyde”) and various oligomers of formaldehydealong with some methanol (typically about 1 wt % to about 15 wt %).

As used herein, the term “hydrogen cyanide” is synonymous with prussicacid, hydrocyanic acid, and all other synonyms of CAS Registry Number200-821-6.

As used herein, the term “glutaraldehyde” is abbreviated “GA” and issynonymous with pentanedial, 1,5-pentanedial, 1,5-pentanedione,diglutaric aldehyde, glutaral, glutardialdehyde, glutaric aciddialdehyde, glutaric dialdehyde, and all other synonyms of CAS RegistryNumber 111-30-8.

As used herein, the term “bisulfite” or “sodium bisulfite” is synonymouswith sulfurous acid sodium salt, sulfurous acid monosodium salt,hydrogen sodium sulfite, hydrogen sulfite sodium, monosodium sulfite,sodium acid sulfite, sodium bisulfite, sodium bisulphate, sodiumhydrogen sulfite, sodium sulfite (NaHSO3), and all other synonyms of CASRegistry Number 7631-90-5.

As used herein, the term “recovering” means isolating, purifying, ortransferring the product formed by the present process. Methods toisolate and purify the product(s) from the reaction mixture are wellknown in the art may include, but are not limited to selectiveprecipitation, crystallization, filtration, reactive solvent extraction,ion exchange, electrodialysis, polymerization, distillation, thermaldecomposition, alcoholysis, column chromatography, and combinationsthereof. In one embodiment, the term “recovering” may also includetransferring the product mixture (typically after filtering out theenzyme catalyst) to another reaction to create one or more additionalproducts. In a preferred embodiment, ion exchange is used to recover theglycolic acid.

As used herein, the terms “enzyme catalyst”, “nitrilase catalyst” or“microbial cell catalyst” refers to a catalyst that is characterized bya nitrilase activity (i.e., comprises at least one polypeptide havingnitrilase activity) for converting glycolonitrile to glycolic acid andammonia. A nitrilase enzyme directly converts a nitrile (preferably, analiphatic nitrile) to the corresponding carboxylic acid, without formingthe corresponding amide as intermediate (see Equation 1). Nitrilasesshare several conserved signature domains known in the art including asignature domain herein referred to as the “catalytic signaturesequence” or “signature sequence”. This region comprises an essentialcysteine residue (e.g., Cys₁₆₄ of SEQ ID NO: 4). As such, polypeptideshaving nitrilase activity can be identified by the existence of thecatalytic domain signature sequence (SEQ ID NO: 1). In a preferredembodiment, the signature sequence is SEQ ID NO: 2. The enzyme catalystmay be in the form of whole microbial cells or permeabilized microbialcells. As used herein, “recycled enzyme catalyst” refers to an enzymecatalyst that is reused as an enzyme catalyst in batch or continuousreactions. Depending on the step in the process of producing or usingthe enzyme catalyst as described herein, the enzyme catalyst may beglutaraldehyde pretreated, immobilized, and cross-linked.

As used herein, the terms “Acidovorax facilis” and “A. facilis” are usedinterchangeably and refer to Acidovorax facilis 72W deposited to theAmerican Type Culture Collection (an international depository authority)having accession number 55746 (“ATCC 55746”). The mutant nitrilasesderived from A. facilis 72W characterized by improved nitrilase activitywhen converting glycolonitrile to glycolic acid have been previouslyreported (see co-owned U.S. Pat. No. 7,198,927). Examples of these A.facilis 72W-derived mutant nitrilases are provided by SEQ ID NOs: 27,29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, and 55.

As used herein, the terms “Escherichia coli” and “E. coli” are usedinterchangeably. Several strains of E. coli suitable for recombinantexpression are described herein including, but not limited to E. coliMG1 655 having international depository number ATCC 47076, E. coli FM5having international depository number ATCC 53911, E. coli W3110 havinginternational depository number ATCC 27325, E. coli MC4100 havinginternational depository number ATCC 35695, and E. coli W1485 havinginternational depository number ATCC 12435. In one embodiment, suitableEscherichia coli strains include E. coli FM5 (ATCC 53911) and E. coliMG1 655 (ATCC 47076).

As used herein, the terms “E. coli SS1001” or “SS1001” refer to atransformed E. coli strain expressing the Acidovorax facilis 72Wnitrilase having ATCC Accession No. PTA-1177 (see U.S. Pat. No.6,870,038; herein incorporated in its entirety by reference). Therecombinantly expressed E. coli SS1001 nitrilase (SEQ ID NO: 57)contains 2 minor sequence changes in comparison to the wild-type 72Wnitrilase sequence (SEQ ID NO: 4). The start codon was changed from GTGto ATG to facilitate recombinant expression and an artifact wasintroduced during cloning that resulted in a single amino acid changenear the C-terminal (Pro367 [CCA]

 Ser [TCA]).

As used herein, the terms “suitable aqueous glycolonitrile reactionmixture” and “suitable aqueous reaction mixture” refer to the materials(including at least one amine protectant) and water in which theglycolonitrile and enzyme catalyst come into contact. The components ofthe suitable aqueous reaction mixture are provided herein and thoseskilled in the art appreciate the range of component variations suitablefor this process.

As used herein, the terms “aqueous ammonium glycolate solution”,“aqueous solution comprising ammonium glycolate”, and “aqueous solutionof ammonium glycolate” will be used to describe an aqueous solutioncomprising ammonium glycolate produced by the enzymatic hydrolysis ofglycolonitrile under typical enzymatic reaction conditions (i.e., a pHrange of about 6 to about 8). The aqueous solution of ammonium glycolatecomprises ammonium glycolate at a concentration of at least about 0.1weight percent (wt %) to about 99 wt % ammonium glycolate. In anotherembodiment, the aqueous solution of ammonium glycolate is comprised ofat least about 10 wt % to about 75 wt % ammonium glycolate. In a furtherembodiment, the aqueous solution of ammonium glycolate is comprised ofat least about 20 wt % to about 50 wt % ammonium glycolate. The pH ofthe aqueous solution of ammonium glycolate can be about 2 to about 12,preferably 5 to about 10, more preferably 6 to about 8. The pH may beadjusted as needed prior to initiating process steps related torecovering glycolic acid (in the form of the acid or salt) from theaqueous ammonium glycolate solution.

As used herein, the terms “catalyst productivity” and “enzyme catalystproductivity” refer to the total amount of product produced per gram ofenzyme catalyst dry cell weight. In the present invention, the enzymecatalyst comprises a nitrilase enzyme (EC 3.5.5.7) and the productformed is glycolic acid and/or ammonium glycolate (depending upon the pHof the reaction). In general, the processes produced pursuant toproducing glycolic acid are conducted under essentially pH neutralconditions so that the glycolic acid produced is predominantly in theform of the corresponding salt of glycolic acid (i.e. ammoniumglycolate). Generally, in batch reactions with catalyst recycle, thecatalyst activity decreases with each recycle reaction (enzymeinactivation).

As used herein, the term “volumetric productivity” refers to thevolumetric production of glycolic acid in the reaction, expressed asgrams of glycolic acid produced per volume of reaction mixture per unitof time. Typically, volumetric productivity is expressed as gramsglycolic acid/L/h.

The term “nitrilase activity” or “specific activity” refers to theenzyme activity per unit mass (for example, milligram) of protein, drycell weight, or bead weight (immobilized catalyst) when convertingglycolonitrile to glycolic acid (or the corresponding ammoniumglycolate). Comparisons in nitrilase activity were measured proportionalto the dry cell weight or bead weight.

As used herein, the term “one unit of enzyme activity” or “one unit ofnitrilase activity” or “U” is defined as the amount of enzyme activityrequired for the production of 1 μmol of glycolic acid product perminute (GLA U/g dry cell weight or bead weight) at a specifiedtemperature (e.g. 25° C.).

As used herein, the terms “relative nitrilase activity”, “improvednitrilase activity”, and “relative improvement in nitrilase activity”refers to the nitrilase activity expressed as a multiple (or fraction)of a reference (control) nitrilase activity. The nitrilases describedherein exhibit a significant improvement in nitrilase activity relativeto the nitrilase activity observed with native Acidovorax facilis 72Wnitrilase. A “significant improvement” in relative nitrilase activity isan improvement of at least 1.5-fold higher nitrilase activity incomparison to the nitrilase activity of a control under identicalreaction conditions. In another embodiment, the improvement is at least2-fold higher nitrilase activity in comparison to the nitrilase activityof the control under identical reaction conditions. In a furtherembodiment, the improvement is at least 4-fold higher nitrilase activityin comparison to the nitrilase activity of the control under identicalreaction conditions.

As used herein, the term “initial reaction rate” is a measurement of therate of conversion of glycolonitrile to glycolic acid under the statedreaction conditions, where the measurement of reaction rate begins uponthe initial addition of glycolonitrile to the reaction mixture, andwhere the reaction rate is measured over a period of time where theconcentration of glycolonitrile remains above ca. 50 millimolar (mM)during the course of the reaction. The reaction rate is measured as thechange in concentration of glycolic acid produced per unit time (e.g.,mole glycolic acid/L/min or mM glycolic acid/hour).

As used herein, the term “improved retention of initial specificactivity” refers to a comparison of a glutaraldehyde pretreated,immobilized and cross-linked enzyme catalyst with a non-glutaraldehydepretreated, immobilized and cross-linked enzyme catalyst, both havingnitrilase activity, during conversion of glycolonitrile to glycolic acidunder the stated reaction conditions, measured as micromoles of glycolicacid produced per minute per g dry cell weight of enzyme catalyst, ormicromoles glycolic acid produced per minute per g immobilized andcrosslinked enzyme catalyst, wherein the specific activity as measuredin a first or “initial” reaction is retained to a greater extent for theglutaraldehyde pretreated immobilized and cross-linked enzyme catalystthan for the non-glutaraldehyde pretreated, immobilized and cross-linkedenzyme catalyst, for one or more subsequent reactions. The most notableimprovement, as described herein, is for the amount of activity retainedfor the reaction immediately following an initial batch reaction,measured in one or more subsequent batch reactions with catalystrecycle. A second notable improvement, as described herein, is for theamount of activity retained during the course of running the reaction ina continuously stirred tank reactor (CSTR), or in a fixed-bed plug flowreactor, or in a fluidized-bed or semi-fluidized bed reactor.

As used herein, the terms “recombinant organism”, “transformed host”,“transformant”, “transgenic organism”, and “transformed microbial host”refer to a host organism having been transformed with heterologous orforeign DNA. The recombinant organisms of the present invention expressforeign coding sequences or genes that encode active nitrilase enzyme.“Transformation” refers to the transfer of a DNA fragment into the hostorganism. The transferred DNA fragment can be chromosomally orextrachromosomally incorporated (i.e., via a vector) into the hostorganism. As used herein, the term “transformation cassette” refers to aspecific fragment of DNA containing a set of genetic elementsconveniently arranged for insertion into a host cell, usually as part ofa plasmid. As used herein, the term “expression cassette” refers to aspecific fragment of DNA containing a set of genetic elementsconveniently arranged for insertion into a host cell, usually as part ofa plasmid that also allows for enhanced gene expression in the host.

As used herein, the terms “nucleic acid fragment” and “nucleic acidmolecule” refer to DNA molecule that may encode an entire gene, codingsequence, and/or regulatory sequences preceding (5′, upstream) orfollowing (3′, downstream) the coding sequence. In one aspect, thepresent nucleic acid molecules encode for polypeptides having nitrilaseactivity.

As used herein, the term “gene” refers to a nucleic acid molecule thatexpresses a specific protein. As used herein, it may or may notincluding regulatory sequences preceding (5′ non-coding sequences) andfollowing (3′ non-coding sequences) the coding sequence. “Chimeric gene”refers to any gene that is not a native gene, comprising regulatory andcoding sequences that are not found together in nature. Accordingly, achimeric gene may comprise regulatory sequences and coding sequencesthat are derived from different sources, or regulatory sequences andcoding sequences derived from the same source, but arranged in a mannerdifferent than that found in nature. “Endogenous gene” refers to anative gene in its natural location in the genome of an organism. A“foreign” gene refers to a gene not normally found in the host organism,but that is introduced into the host organism by gene transfer. Foreigngenes can comprise native genes inserted into a non-native organism, orchimeric genes. A “transgene” is a gene that has been introduced intothe genome by a transformation procedure.

As used herein, the term “coding sequence” refers to a DNA sequence thatcodes for a specific amino acid sequence. As used herein, “suitableregulatory sequences” refer to nucleotide sequences located upstream (5′non-coding sequences), within, or downstream (3′ non-coding sequences)of a coding sequence, and which influence the transcription, RNAprocessing or stability, or translation of the associated codingsequence. Regulatory sequences may include promoters, translation leadersequences, introns, polyadenylation recognition sequences, RNAprocessing sites, effector binding sites, and stem-loop structures.

“Promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. Promoters that cause a gene to beexpressed in most cell types at most times or under most environmentalconditions are commonly referred to as “constitutive promoters”.Promoters that cause a gene to be expressed only in the presence of aparticular compound or environmental condition are commonly referred toas “inducible promoters”. Since in most cases the exact boundaries ofregulatory sequences have not been completely defined, DNA fragments ofdifferent lengths may have identical promoter activity.

As used herein, the term “operably linked” refers to the association ofnucleic acid sequences on a single nucleic acid molecule so that thefunction of one sequence is affected by the other. For example, apromoter is operably linked with a coding sequence when it is capable ofaffecting the expression of that coding sequence (i.e., that the codingsequence is under the transcriptional control of the promoter). Codingsequences can be operably linked to regulatory sequences in sense orantisense orientation.

As used herein, the term “3′ non-coding sequences” refers to DNAsequences located downstream of a coding sequence and includepolyadenylation recognition sequences (normally limited to eukaryotes)and other sequences encoding regulatory signals capable of affectingmRNA processing or gene expression. The polyadenylation signal (normallylimited to eukaryotes) is usually characterized by affecting theaddition of polyadenylic acid tracts to the 3′ end of the mRNAprecursor.

The skilled artisan is well aware of the “codon-bias” exhibited by aspecific host cell in using nucleotide codons to specify a given aminoacid. Therefore, when synthesizing a gene for improved expression in ahost cell, it is desirable to design the gene such that its codon usagereflects the preferred codon bias of the host cell. A survey of genesderived from the host cell where sequence information is available candetermine its codon bias. Codon-optimization is well known in the artand has been described for various systems including, but not limited toyeast (Outchkourov et al., Protein Expr Purif, 24(1):18-24 (2002)) andE. coli (Feng et al., Biochemistry, 39(50):15399-15409 (2000)).

Enzyme Catalysts having Nitrilase Activity

All nitrilases (EC 3.5.5.7) share a conserved catalytic triad (Glu, Lys,and Cys) (Chauhan et al., Appl. Microbiol. Biotechnol. 61:118-122(2003); Pace, H. and Brenner, C., Genome Biol. [online computer file]2(1):reviews0001.1-0001.9 (2001)). All known nitrilases have anucleophilic cysteine in the enzyme active site (Cowan et al.,Extremophiles, 2:207-216 (1998); Pace, H. and Brenner, C., supra; andChauhan et al., supra) and all are susceptible to inactivation by thiolreagents (1.0 mM concentrations of copper chloride, silver nitrate,mercuric acetate, or ferric chloride each produced major decreases in A.facilis 72W nitrilase enzyme activity). Cysteine residues are alsocapable of being irreversibly oxidized to sulfinic acids, resulting in aloss of enzyme activity. Despite the sensitivity of nitrilase enzymes tovarious inactivating mechanisms, immobilized A. facilis 72W cells arerobust, capable of retaining much of their nitrilase activity afternumerous recycle reactions (U.S. Pat. No. 6,870,038; U.S. Pat. No.7,148,051; U.S. Pat. No. 7,198,927; and Chauhan et al., supra).Nitrilase catalysts derived from the A. facilis 72W nitrilase also beenshown to catalyze the conversion of α-hydroxynitriles (i.e.,glycolonitrile) to α-hydroxycarboxylic acids (i.e., glycolic acid) (seeU.S. Pat. No. 6,383,786; U.S. Pat. No. 6,416,980; and U.S. Pat. No.7,198,927).

Sequence comparisons of the A. facilis 72W nitrilase to other bacterialnitrilases have been reported (U.S. Pat. No. 6,870,038; Chauhan et al.,supra). The 72W nitrilase has several conserved signature domainsincluding a 16-amino acid region near the amino terminus (amino acidresidues 40-55 of SEQ ID NO: 4) and a 12 amino acid catalytic region(amino acid residues 160-171 of SEQ ID NO: 4) containing the essentialcysteine residue. This essential cysteine residue (Cys₁₆₄ of SEQ ID NO:4), along with conserved glutamic acid (Glu₄₈ of SEQ ID NO:4) and lysineresidues (Lys₁₃₀ of SEQ ID NO:4), form the catalytic triad motif foundin all nitrilases (Pace, H., and Brenner, C., supra).

The regions surrounding each of the catalytic triad residues are highlyconserved, especially the region surrounding the catalytic cysteineresidue. The essential catalytic cysteine residue is located with ahighly conserved region referred to as the “catalytic signature motif”or “signature motif”. As such, the present process is useful forprotecting the enzymatic activity of any nitrilase comprising thecatalytic signature motif defined by Formula 1 (bold indicates strictlyconserved amino acid residues, italicized residues are those thatexhibit minimal variability [i.e. minimal variation of 3 or fewer aminoacid residues], the catalytic cysteine residue is underlined):

Formula 1 (SEQ ID NO: 1) Gly-Xaa ₁-Xaa ₂-Xaa₃- Cys -Trp-Glu-Xaa₄-Xaa₅-Xaa₆- Xaa ₇-Xaa ₈.

wherein

-   -   Xaa₁=Ala or Gly;    -   Xaa₂=Leu, Val, or Ala;    -   Xaa₃=Ala, Asn, lie, Cys, Val, or Gln;    -   Xaa₄=His or Asn;    -   Xaa₅=Leu, Tyr, Phe, Ala, Met, Lys, Val, Thr, or Arg;    -   Xaa₆=Asn, Gln, Met, Leu, or Ser;    -   Xaa₇=Pro or Thr; and    -   Xaa₈=Leu or Val.

In a preferred embodiment, the nitrilase signature motif of Formula 1 isXaa₁=Ala or Gly; Xaa₂=Leu; Xaa₃=Ala, Asn, lie, Cys, Val, or Gln;Xaa₄=His; Xaa₅=Leu, Tyr, Phe, Ala, Met, Lys, Val, Thr or Arg; Xaa₆=Ser,Gln, Asn, or Met; Xaa₇=Pro; and Xaa₈=Leu; resulting in the catalyticsignature motif represented by the following:

(SEQ ID NO: 2) Gly-Xaa₁-Leu-Xaa₃-Cys-Trp-Glu-His-Xaa₅-Xaa₆- Pro-Leu

Examples of nitrilases, including the sequences and position of thecorresponding catalytic signature motif sequence, are provided in Table1.

TABLE 1 Conserved Catalytic Cysteine Region- Catalytic Signature MotifsGenBank ® Sequence Nitrilase Accession Amino Acid of Signature MotifSource Number SEQ ID NO. (amino acid residue positions) AcidovoraxFacilis ABD98457.1 4 GGLNCWEHFQPL 72W (160-171) Alcaligenes faecalisBAA02684.1 5 GALCCWEHLSPL JM3 (159-170) Rhodococcus Q03217 6GALNCWEHFQTL rhodochrous J1 (161-172) Rhodococcus Q02068 7 GGLNCWEHFQPLrhodochrous K22 (166-177) Nocardia sp. C-14-1 AAX18182.1 8 GGLNCWEHFQPL(154-165) Bordetella NP_887662.1 9 GAVVCWENYMPL bronchiseptica RB50(161-172) Arabidopsis thaliana AAB60275.1 10 GAAICWENRMPL AAA19627.1(175-186) Synechococcus YP_399857.1 11 GALACWEHYNPL elongatus PCC 7942(157-168) Synechococcus YP_171411.1 12 GALACWEHYNPL elongatus PCC 6301(157-168) Synechocystis sp. NP_442646.1 13 GALACWEHYNPL PCC 6803(165-176) Pseudomonas YP_6090481.1 14 GAAVCWENYMPL entomophila L48(161-172) Zymomonas moblis YP_162942.1 15 GAAICWENYMPV (161-172)Bacillus sp. OxB-1 BAA90460.1 16 GGLQCWEHFLPL (158-169) ComamonasAAA82085.1 17 GGLQCWEHALPL testosteroni (159-170) Synechococcus sp.YP_381420.1 18 GALACWEHYNPL CC9605 (156-167) Pseudomonas YP_260015.1 19GAVICWENMMPL fluorescens Pf-5 (161-172) Nocardia farcinica YP_119480.120 GALCCWEHLQPL IFM 10152 (159-170) Alcaligenes faecalis AAY06506.1 21GALCCWEHLSPL 1650 (159-170) Pseudomonas AAY35081.1 22 GALCCWEHLQPLsyringae pv. (157-168) syringae B728a Bradyrhizobium sp. ZP_00859948.123 GALCCWEHLQPL BTAil (163-174) Rhodococcus CAC88237 24 GALNCWEHFQTLrhodochrous (161-172) NCIMB 11216 Rhodococcus N/A 25 GALNCWEHFQTLrhodochrous ATCC (161-172) 39484 ™

In one embodiment, the nitrilase catalyst comprises a polypeptide havingnitrilase activity isolated from a genera selected from the groupconsisting of Acidovorax, Rhodococcus, Nocardia, Bacillus, andAlcaligenes. In one embodiment, the nitrilase catalyst comprises apolypeptide having nitrilase activity isolated from a genera selectedfrom the group consisting of Acidovorax and Rhodococcus.

In another embodiment, the polypeptide having nitrilase activity isderived from Acidovorax facilis 72W (ATCC 55746) or a polypeptide(having nitrilase activity) that is substantially similar to theAcidovorax facilis 72W nitrilase (SEQ ID NO: 4) or the A. facilis 72Wderived enzyme represented by SEQ ID NO: 51.

In one embodiment, the nitrilase catalyst is a microbial host celltransformed to express at least one polypeptide having nitrilaseactivity. In one embodiment the transformed host cell is selected fromthe group consisting of Comamonas sp., Corynebacterium sp.,Brevibacterium sp., Rhodococcus sp., Azotobacter sp., Citrobacter sp.,Enterobacter sp., Clostridium sp., Klebsiella sp., Salmonella sp.,Lactobacillus sp., Aspergillus sp., Saccharomyces sp., Yarrowia sp.,Zygosaccharomyces sp., Pichia sp., Kluyveromyces sp., Candida sp.,Hansenula sp., Dunaliella sp., Debaryomyces sp., Mucor sp., Torulopsissp., Methylobacteria sp., Bacillus sp., Escherichia sp., Pseudomonassp., Rhizobium sp., and Streptomyces sp. In a preferred embodiment, themicrobial host cell is selected from the group consisting of Bacillussp., Pseudomonas sp., and Escherichia sp. In a preferred embodiment, thecatalyst is an Escherichia coli host cell recombinantly expressing oneor more of the polypeptides having nitrilase activity.

In another embodiment, the nitrilase catalyst comprises a polypeptidehaving nitrilase activity wherein said polypeptide having nitrilaseactivity has at least 60% identity to SEQ ID NO: 51, preferably at least70% identity to SEQ ID NO: 51, even more preferably at least 80%identity to SEQ ID NO: 51, yet even more preferably at least 90%identity to SEQ ID NO: 51, and most preferably at least 95% identity toSEQ ID NO: 51.

Working examples of several catalysts having nitrilase activity derivedfrom various sources are described herein, including a catalyst derivedfrom the A. facilis 72W nitrilase. Various mutants derived from theAcidovorax facilis 72W nitrilase enzyme have been reported in the art(U.S. Pat. No. 7,148,051 and U.S. Pat. No. 7,198,927).

In one embodiment, the polypeptide having nitrilase activity is selectedfrom the group consisting of SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 47, 49, 51, 53, 55, and 57. In another embodiment,the polypeptide having nitrilase activity is selected from the groupconsisting of 4, 24, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49,51, 53, 55, and 57. In another embodiment, the polypeptide havingnitrilase activity is selected from the group consisting of 4, 27, 29,31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, and 57. n anotherembodiment, the polypeptide having nitrilase activity is selected fromthe group consisting of 4, 24, 25, and 51. In another embodiment, thenitrilase catalyst comprises the polypeptide of SEQ ID NO: 51.

Acidovorax Facilis 72W (ATCC 55746) Nitrilase

The A. facilis 72W nitrilase (EC 3.5.5.1) is a robust catalyst forproducing carboxylic acids from aliphatic or aromatic nitriles (WO01/75077; U.S. Pat. No. 6,870,038; and Chauhan et al., supra). It hasalso been shown to catalyze the conversion of α-hydroxynitriles (i.e.,glycolonitrile) to α-hydroxycarboxylic acids (i.e., glycolic acid) (seeU.S. Pat. No. 6,383,786 and U.S. Pat. No. 6,416,980). However, nitrilasecatalysts having improved nitrilase activity and/or stability (relativeto the A. facilis 72W nitrilase) when converting glycolonitrile toglycolic acid would reduce the cost of manufacturing glycolic acid. Assuch, a method of producing glycolic acid using an improved nitrilasecatalyst is useful to reduce the cost of manufacturing glycolic acid,however A. facilis 72W nitrilase is an enzyme catalyst for purposes ofthe processes herein, as well as said improved nitrilases described indetail above.

Industrial Production of the Enzyme Catalyst

Where commercial production of the enzyme catalysts described herein isdesired, a variety of culture methodologies may be used. Fermentationruns may be conducted in batch, fed-batch, or continuous mode, methodswell-known in the art (Thomas D. Brock in Biotechnology: A Textbook ofIndustrial Microbiology, Second Edition (1989) Sinauer Associates, Inc.,Sunderland, Mass., (1989); Deshpande, Mukund V., Appl. Biochem.Biotechnol. 36(3): 227-234 (1992)).

A classical batch culturing method is a closed system where thecomposition of the media is set at the beginning of the culture and notsubject to artificial alterations during the culturing process. Thus, atthe beginning of the culturing process the media is inoculated with thedesired organism or organisms and growth or metabolic activity ispermitted to occur adding nothing to the system. Typically, however, a“batch” culture is batch with respect to the addition of carbon sourceand attempts are often made at controlling factors such as pH and oxygenconcentration. In batch systems the metabolite and biomass compositionsof the system change constantly up to the time the culture isterminated. Within batch cultures cells moderate through a static lagphase to a high growth log phase and finally to a stationary phase wheregrowth rate is diminished or halted. If untreated, cells in thestationary phase will eventually die. Cells in log phase are oftenresponsible for the bulk of production of end product or intermediate insome systems. Stationary or post-exponential phase production can beobtained in other systems.

A variation on the standard batch system is the Fed-Batch system.Fed-Batch culture processes are also suitable in the present inventionand comprise a typical batch system with the exception that thesubstrate is added in increments as the culture progresses. Fed-Batchsystems are useful when catabolite repression is apt to inhibit themetabolism of the cells and where it is desirable to have limitedamounts of substrate in the media. Measurement of the actual substrateconcentration in Fed-Batch systems is difficult and is thereforeestimated on the basis of the changes of measurable factors such as pH,dissolved oxygen, and the partial pressure of waste gases such as CO₂.Batch and Fed-Batch culturing methods are common and well known in theart and examples may be found in Brock (supra) and Deshpande (supra).

Commercial production of the present enzyme catalysts having nitrilaseactivity may also be accomplished with a continuous culture. Continuouscultures are an open system where a defined culture media is addedcontinuously to a bioreactor and an equal amount of conditioned media isremoved simultaneously for processing. Continuous cultures generallymaintain the cells at a constant high-liquid-phase density where cellsare primarily in log phase growth. Alternatively, continuous culture maybe practiced with immobilized cells where carbon and nutrients arecontinuously added and valuable products, by-products or waste productsare continuously removed from the cell mass. Cell immobilization may beperformed using a wide range of solid supports composed of naturaland/or synthetic materials.

Continuous or semi-continuous culture allows for the modulation of onefactor or any number of factors that affect cell growth or end cellconcentration. For example, one method will maintain a limiting nutrientsuch as the carbon source or nitrogen level at a fixed rate and allowall other parameters to moderate. In other systems a number of factorsaffecting growth can be altered continuously while the cellconcentration, measured by media turbidity, is kept constant. Continuoussystems strive to maintain steady-state growth conditions and thus thecell loss due to media being drawn off must be balanced against the cellgrowth rate in the culture. Methods of modulating nutrients and growthfactors for continuous culture processes, as well as techniques formaximizing the rate of cell formation, are well known in the art ofindustrial microbiology and a variety of methods are detailed by Brock(supra).

Fermentation media in the present invention must contain suitable carbonsubstrates. Suitable substrates may include, but are not limited tomonosaccharides such as glucose and fructose, disaccharides such aslactose or sucrose, polysaccharides such as starch or cellulose ormixtures thereof, and unpurified mixtures from renewable feedstocks suchas cheese whey permeate, cornsteep liquor, sugar beet molasses, andbarley malt. Hence, it is contemplated that the source of carbonutilized in the present invention may encompass a wide variety ofcarbon-containing substrates and will only be limited by the choice oforganism.

Glutaraldehyde Pretreatment of the Enzyme Catalyst Prior toImmobilization

Treatment of an enzyme catalyst fermentation culture with glutaraldehydecan be a convenient way to kill the microbes in the culture, thusavoiding containment and safety issues for handling, storage andtransportation associated with live recombinant cultures. It has nowbeen discovered that pretreatment with glutaraldehyde, or glutaraldehydepretreatment followed by bisulfite treatment, can preserve nitrilaseactivity in cells in suspension and in an immobilized form.

Preservation of nitrilase activity with glutaraldehyde pretreatment ofan enzyme catalyst is affected by time, temperature, glutaraldehydeconcentration, pH and the concentration of inhibitory products likeammonia and other amines (e.g., amino acids and peptides) in the mediathat interact with glutaraldehyde. A preferred glutaraldehydepretreatment method treats cells from high-density fermentation (100-150OD₅₅₀) with 5-10 wt % glutaraldehyde in water that is preferablydelivered with adequate mixing at 50 mg to 500 mg glutaraldehyde/L-min,more preferably delivered with adequate mixing at 50 mg to 200 mgglutaraldehyde/L-min, most preferably delivered with adequate mixing at50 mg to 100 mg glutaraldehyde/L-min, resulting in a final concentrationof about 3 g to about 5 g glutaraldehyde/L (about 0.025 g to about 0.042g glutaraldehyde per OD₅₅₀), more preferably about 3.6 g to about 5 gglutaraldehyde/L (about 0.030 g to about 0.042 g glutaraldehyde perOD₅₅₀). The glutaraldehyde pretreated culture may be held in thefermenter for about 1 to 5 hours. A 10 wt % solution of sodium bisulfitein water is then optionally added at 1 g/L to inactivate the residualglutaraldehyde.

The preferred pH for the glutaraldehyde pretreatment of the enzymecatalyst in the fermentation broth or cell suspension is from pH 5.0 to9.0, more preferably from pH 5.0 to 8.0, even more preferably from pH5.0 to 7.0, still more preferably pH 5.0 to 6.0, and most preferably pH5.0 to 5.5. The residual glutaraldehyde concentration afterglutaraldehyde pretreatment is typically low, in the range of 10-200ppm, and can be inactivated as stated above, with the addition of sodiumbisulfite to a final concentration of about 1 g/L. Glutaraldehyde andbisulfite pretreatment were found to have no significant detrimentaleffect on the nitrilase activity. The glutaraldehyde or glutaraldehyde/bisulfite pretreated cell suspension is optionally chilled to 5-10° C.,and optionally washed (by concentration and re-dilution of the cellsuspension or fermentation broth) with water or an appropriate storagebuffer to remove residual bisulfite and unreacted glutaraldehyde.

Immobilization of Glutaraldehyde Pretreated Enzyme Catalyst and ChemicalCross-linking

Methods for the immobilization of enzyme catalysts have been widelyreported and are well known to those skilled in the art (Methods inBiotechnology, Vol. 1: Immobilization of Enzymes and Cells; Gordon F.Bickerstaff, Editor; Humana Press, Totowa, N.J., USA; 1997). Theimmobilization of the A. facilis 72W nitrilase catalyst has also beenpreviously reported (U.S. Pat. No. 6,870,038).

Further, a method for immobilization in carrageenan and subsequentglutaraldehyde/polyethylenimine cross-linking of the immobilized enzymecatalyst follows (and as disclosed in U.S. Pat. No. 6,870,038, and asdescribed in detail in U.S. Pat. No. 6,551,804 B, herein incorporated byreference), however, one of ordinary skill in the art would recognizeand readily apply variations to accomplish immobilization andcross-linking. Said variations are contemplated herein and are withinthe scope of the instant process. Further, the amounts or concentrationsof components used for immobilization and chemical cross-linking willvary depending on the amount and type of enzyme catalyst andfermentative production of enzyme catalyst. One of ordinary skill in theart would recognize these factors and adjust the immobilization andchemical cross-linking procedures accordingly. With regard tocross-linking with glutaraldehyde and polyethylenimine, U.S. Pat. No.6,551,804 (supra), describes the processes and procedures for chemicallycross-linking alginate immobilized cells. Said description applies herefor carrageenan immobilized cells as well.

Hydrolysis of Glycolonitrile to Glycolic Acid Using a Nitrilase Catalyst

The enzymatic conversion of glycolonitrile to glycolic acid (in the formof the acid and/or the corresponding ammonium salt) may be performed bycontacting an enzyme catalyst, immobilized enzyme catalyst, orcross-linked immobilized enzyme catalyst having nitrilase activity undersuitable reaction conditions as described below (i.e. in an aqueousreaction mixture at certain pH range, temperatures, concentrations,etc.). In one embodiment, whole recombinant microbial cells areimmobilized in carrageenan, cross-linked, and the resulting enzymecatalyst used directly for the conversion of glycolonitrile to glycolicacid, or unimmobilized cells can be maintained separately from the bulkreaction mixture using hollow-fiber membrane cartridges orultrafiltration membranes. In a second embodiment, whole recombinantmicrobial cells are immobilized in polyacrylamide gel, and the resultingenzyme catalyst used directly for the conversion of glycolonitrile toglycolic acid.

The concentration of enzyme catalyst in an aqueous reaction mixturedepends on the specific activity of the enzyme catalyst and is chosen toobtain the desired rate of reaction. The wet cell weight of themicrobial cells used as catalyst in hydrolysis reactions typicallyranges from 0.001 grams to 0.250 grams of wet cells per mL of totalreaction volume, preferably from 0.002 grams to 0.050 grams of wet cellsper mL. The indicated wt % of wet cells per volume of total reactionvolume may be present in the reaction mixture in the form of animmobilized enzyme catalyst prepared as previously described (supra),where the weight of wet cells as a percentage of the total weight of theimmobilized enzyme catalyst is known from the method of preparation ofthe immobilized enzyme catalyst.

The temperature of the glycolonitrile hydrolysis reaction is chosen tocontrol both the reaction rate and the stability of the enzyme catalystactivity. The temperature of the reaction may range from just above thefreezing point of the reaction mixture (approximately 0° C.) to about65° C., with a preferred range of reaction temperature of from about 5°C. to about 35° C. An enzyme catalyst suspension may be prepared bysuspending the immobilized cells in distilled water, or in a aqueoussolution of a buffer which will maintain the initial pH of the reactionbetween about 5.0 and about 10.0, preferably between about 5.5 and about8.0, more preferably between about 5.5 and about 7.7, and mostpreferably about 6.0 to about 7.7. As the reaction proceeds, the pH ofthe reaction mixture may change due to the formation of an ammonium saltof the carboxylic acid from the corresponding nitrile functionality. Thereaction can be run to complete conversion of glycolonitrile with no pHcontrol, or a suitable acid or base can be added over the course of thereaction to maintain the desired pH.

Glycolonitrile was found to be completely miscible with water in allproportions at 25° C. In cases where reaction conditions are chosen suchthat the solubility of the substrate (i.e., an α-hydroxynitrile) is alsodependent on the temperature of the solution and/or the saltconcentration (buffer or product glycolic acid ammonium salt, also knownas ammonium glycolate) in the aqueous phase, the reaction mixture mayinitially be composed of two phases: an aqueous phase containing theenzyme catalyst and dissolved α-hydroxynitrile, and an organic phase(the undissolved α-hydroxynitrile). As the reaction progresses, theα-hydroxynitrile dissolves into the aqueous phase, and eventually asingle phase product mixture is obtained. The reaction may also be runby adding the α-hydroxynitrile to the reaction mixture at a rateapproximately equal to the enzymatic hydrolysis reaction rate, therebymaintaining a single-phase aqueous reaction mixture, and avoiding thepotential problem of substrate inhibition of the enzyme at high startingmaterial concentrations.

Glycolic acid may exist in the product mixture as a mixture of theprotonated carboxylic acid and/or its corresponding ammonium salt(dependent on the pH of the product mixture; pKa of glycolic acid isabout 3.83), and may additionally be present as a salt of the carboxylicacid with any buffer that may additionally be present in the productmixture. Typically, the glycolic acid produced is primarily in the formof the ammonium salt (pH of the glycolonitrile hydrolysis reaction istypically between about 5.5 and about 7.7). The glycolic acid productmay be isolated from the reaction mixture as the protonated carboxylicacid, or as a salt of the carboxylic acid, as desired.

The final concentration of glycolic acid in the product mixture atcomplete conversion of glycolonitrile may range from 0.001 M to thesolubility limit of the glycolic acid product. In one embodiment, theconcentration of glycolic acid will range from about 0.10 M to about 5.0M. In another embodiment, the concentration of glycolic acid will rangefrom about 0.2 M to about 3.0 M.

Glycolic acid may be recovered in the form of the acid or correspondingsalt using a variety of techniques including, but not limited to ionexchange, electrodialysis, reactive solvent extraction, polymerization,thermal decomposition, alcoholysis, and combinations thereof.

Further, when an amount, concentration, or other value or parameter isgiven either as a range, preferred range, or a list of upper preferablevalues and lower preferable values, this is to be understood asspecifically disclosing all ranges formed from any pair of any upperrange limit or preferred value and any lower range limit or preferredvalue, regardless of whether ranges are separately disclosed. Where arange of numerical values is recited herein, unless otherwise stated,the range is intended to include the endpoints thereof, and all integersand fractions within the range. It is not intended that the scope of theinvention be limited to the specific values recited when defining arange.

General Methods

The following examples are provided to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus may be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Materials and methods suitable for the maintenance and growth ofbacterial cultures are well known in the art. Techniques suitable foruse in the following examples may be found as set out in Manual ofMethods for General Bacteriology (1994) (Phillipp Gerhardt, R. G. E.Murray, Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R.Krieg and G. Briggs Phillips, eds.), American Society for Microbiology,Washington, D.C.) or by Thomas D. Brock, in Biotechnology: A Textbook ofIndustrial Microbiology, (1989) Second Edition, (Sinauer Associates,Inc., Sunderland, Mass.). Methods to immobilize enzymatic catalysts canbe found in Bickerstaff, G. F., supra).

Procedures required for genomic DNA preparation, PCR amplification, DNAmodifications by endo- and exo-nucleases for generating desired ends forcloning of DNA, ligations, and bacterial transformation are well knownin the art. Standard recombinant DNA and molecular cloning techniquesused here are well known in the art and are described by Maniatis,supra; and by T. J. Silhavy, M. L. Bennan, and L. W. Enquist,Experiments with Gene Fusions, (1984) Cold Spring Harbor LaboratoryPress, Cold Spring, N.Y.; and by Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, (1994-1998) John Wiley & Sons, Inc., NewYork.

All reagents and materials were obtained from Aldrich Chemicals(Milwaukee, Wis.), DIFCO Laboratories (Detroit, Mich.), GIBCO/BRL(Gaithersburg, Md.), or Sigma/Aldrich Chemical Company (St. Louis, Mo.)unless otherwise specified.

The abbreviations in the specification correspond to units of measure,techniques, properties, or compounds as follows: “sec” means second(s),“min” means minute(s), “h” or “hr” means hour(s), “d” means density ing/mL, “μL” means microliters, “mL” means milliliters, “L” means liters,“mM” means millimolar, “M” means molar, “mmol” means millimole(s), “wt”means weight, “wt %” means weight percent, “g” means grams, “μg” meansmicrograms, HPLC” means high performance liquid chromatography, “O.D.”means optical density at the designated wavelength, “dcw” means dry cellweight, “U” means units of nitrilase activity, “EDTA” meansethylenediaminetetraacetic acid, and “DTT” means dithiothreitol. One Uof nitrilase activity corresponds to the hydrolysis of 1 μmolglycolonitrile/min.

Analytical Methodology

HPLC Analysis

Unless otherwise noted, the following HPLC method was used. The reactionproduct mixtures were analyzed by the following HPLC method. Aliquots(0.01 mL) of the reaction mixture were added to 1.50 mL of water, andanalyzed by HPLC (HPX 87H column, 30 cm×7.8 mm; 0.01 N H₂SO₄ mobilephase; 1.0 mL/min flow at 50° C.; 10 μL injection volume; RI detector,20 min analysis time). The method was calibrated for glycolonitrile at aseries of concentrations using commercially available glycolonitrilepurchased from Aldrich.

Quantitative ¹³C NMR Analysis

Quantitative ¹³C NMR spectra were obtained using a Varian Unity Inovaspectrometer (Varian, Inc., Palo Alto, Calif.) operating at 400 MHz.Samples were prepared by taking 3.0 mL of the reaction product alongwith 0.5 mL of D₂O in a 10 mm NMR tube. ¹³C NMR spectra were typicallyacquired using a spectral width of 26 KHz with the transmitter locatedat 100 ppm, 128K points, and a 90-degree pulse (pw90=10.7 microsecondsat a transmitter power of 56 db). The longest 13C T1 (23 sec) wasassociated with the GLN nitrile carbon, and the total recycle time wasset greater than ten times this value (recycle delay d1=240 sec,acquisition time at=2.52 sec). Signal averaging of 360 scans gave atotal experiment time of 26.3 hours. The Nuclear Overhauser Enhancement(NOE) was suppressed by gating on the Waltz-modulated 1H decoupling onlyduring the acquisition time (at).

EXAMPLE 1 Fermentation of E. coli MG1655/ISW138-168V

Seed cultures of E. coli MG1655/pSW138-168V were grown in 500 mL LBmedia supplemented with 0.1 mg ampicillin per mL for 6-10 h (OD₅₅₀=1-2)at 30° C. with shaking (300 rpm) prior to inoculation of the fermentor.Growth of E. coli MG1655/pSW138-168V nitrilase strain was in 14-L BraunBiostat C fermenters (B. Braun Biotech International Gmbh, Melsungen,Germany) using mineral medium with glucose, ammonia, and salts, andlactose was used for induction. Pre-sterilization fermenter media (7.5L) is described in Table 2. Post-sterilization additions includefilter-sterilized trace elements (Table 3), 0.1 mg ampicillin per mL, 2g casamino acids (Difco) per L, 4 g glucose per L, and 500 mL seedculture.

Fermentation set points are described in Table 4. NH₄OH (40% w/v) andH₃PO₄ (20% w/v) were used for pH control. The dissolved oxygenconcentration was controlled at 25% of air saturation with the agitationset to rise first with increase in oxygen demand, with aeration tofollow. The fermentation feed protocol used with lactose induction isgiven in Table 5. Glucose feed rates were reduced if glucose accumulatedabove 5 g/L. After 40-56 h, the fermentation broth was chilled to 5-10°C. and the cells harvested by centrifugation. Cell paste was frozen andstored at −70° C. The cell paste was designated as NIT 60 (1910 GLN U/gdcw).

TABLE 2 Fermentation media, pre-sterilization. (NH₄)₂SO₄ 5.0 g/L K₂HPO₄4.0 g/L KH₂PO₄ 3.5 g/L MgSO₄*7H₂O 0.6 g/L Na₃Citrate*2H₂O 1.0 g/L NZAmine AS (Quest) 2.5 g/L Antifoam-Biospumex 153K 0.25 ml/L

TABLE 3 Fermentation trace elements Concentration Citric acid  10 g/LCaCl₂*2H₂O 1.5 g/L FeSO₄*7H₂O   5 g/L ZnSO₄*7H₂O 0.39 g/L  CuSO₄*5H₂O0.38 g/L  CoCl₂*6H₂O 0.2 g/L MnCl₂*4H₂O 0.3 g/L

TABLE 4 Fermentation set points Initial Set-Point Minimum MaximumStirrer (rpm) 400 400 1000 Airflow (slpm) 2 2 10 pH 6.8 6.8 6.8 Pressure(kPa) 0.5 0.5 0.5 DO 25% 25% 25% Temperature ° C. 30 30 30

TABLE 5 Fermentation feed protocol used with lactose induction Feed RateEFT (h) (g/min) Substrate 0 0 Glucose (batched) 5 0.27 Glucose (50% w/w)14 1.3 Lactose (25% w/w)

EXAMPLE 2 Immobilization of E. coli MG1655/INM18-168V inGA/PEI-Cross-Linked Carrageenan Beads

With rapid stirring, 12 g of carrageenan (FMC GP911) was slowly added to228 g deionized distilled water at 50° C., the resulting mixture heatedto 80° C. until the carrageenan was completely dissolved, and theresulting solution cooled with stirring to 52° C. In a separate beakerequipped with stir bar, 83.2 g of frozen E. coli MG1655/pNM18-168V cells(25.2% dcw) were added to 84.8 g of 0.35 M Na₂HPO₄ (pH 7.3) at ca. 25°C. and mixed until the cells were suspended, then a deoxyribonuclease Isolution (10 μL of 12,500 U/mL DNase (Sigma)/100 mL of cell suspension)was added. The cell suspension was filtered consecutively through a 230micron and 140 micron Nupro TF strainer element filter, and heated withstirring to 50° C. With stirring, 160.0 g of E. coli MG1655/pNM18-168Vcell suspension at 50° C. was added to the carrageenan solution at 52°C., and the resulting cell/carrageenan suspension was pumped through anelectrically-heated 20 gauge needle at 47° C. and dripped into 0.25 MKHCO₃ (pH=7.3) with stirring at ca. 37-38° C.); the flow rate throughthe needle was set at 5-8 mL/min. The resulting beads were allowed toharden in this same buffer for 1 h at room temperature with stirring,and were stored in 0.25 M potassium bicarbonate (pH 7.3).

Chemical cross-linking of the immobilized cell/carrageenan beads wasperformed by addition of 0.5 g of 25% glutaraldehyde (GA) in water(Sigma M 752-07) to 20 g beads suspended in 48 mL of 0.25 M potassiumbicarbonate (pH 7.3), and stirring for 1 h at room temperature. To thesuspension of beads was then added 2.0 g of 12.5 wt % polyethylenimine(PEI, BASF LUPASOL PS) in water, and the bead suspension stirred for anadditional 18 h at room temperature. The GA/PEI-cross-linked beads wererecovered from the suspension, stirred twice for 15 min in 48 mL of 0.25M potassium bicarbonate (pH 7.3), then stored in 1.0 M ammoniumbicarbonate (pH 7.3) at 5° C. Prior to use as catalyst for conversion ofglycolonitrile to glycolic acid (as the ammonium salt), the beads werewashed twice for 15 min with 180 mL of 0.1 M ammonium glycolate (pH 7.3)at room temperature to remove the 1.0 M ammonium bicarbonate (pH 7.3)storage buffer. The resulting immobilized cell catalyst was identifiedas immobilized NIT 60.

EXAMPLE 3 Pretreatment of E. coli MG1655/ISW138-168V with GlutaraldehydePrior to Immobilization

A 200-L fermentation was performed to produce a broth containing E. coliMG1655/pSW138-168V cells that were subsequently pretreated withglutaraldehyde in-situ prior to immobilization. A pre-seed culture wasfirst prepared by charging a 2-L shake flask with 0.5 L seed mediumcontaining yeast extract (Ambrex 695, 5.0 g/L), K₂HPO₄ (10.0 g/L),KH₂PO₄ (7.0 g/L), sodium citrate dihydrate (1.0 g/L), (NH₄)₂SO₄ (4.0g/L), MgSO₄ heptahydrate (1.0 g/L) and ferric ammonium citrate (0.10g/L). The pH of the medium was adjusted to 6.8 and the medium wassterilized in the flask. Post sterilization additions included glucose(10 mL, 50 wt %) and 1 mL ampicillin (25 mg/mL). The pre-seed medium wasinoculated with a 1-mL frozen stock culture of E. coliMG1655/pSW138-168V in 20% glycerol, and cultivated at 35° C. and 300rpm. The seed culture was transferred at ca. 2 OD₅₅₀ to a 14L seedfermentor (Braun) with 8 L of medium containing KH₂PO₄ (3.50 g/L), FeSO₄heptahydrate (0.05 g/L), MgSO₄ heptahydrate (2.0 g/L), sodium citratedihydrate (1.90 g/L), yeast extract (Ambrex 695, 5.0 g/L), Biospumexl53K antifoam (0.25 mL/L, Cognis Corporation), NaCl (1.0 g/L), CaCl₂dihydrate (10 g/L), and NIT trace elements solution (10 mL/L). The traceelements solution contained citric acid monohydrate (10 g/L), MnSO₄hydrate (2 g/L), NaCl (2 g/L), FeSO₄ heptahydrate (0.5 g/L), ZnSO₄heptahydrate (0.2 g/L), CuSO₄ pentahydrate (0.02 g/L) and NaMoO₄dihydrate (0.02 g/L). Post sterilization additions included 120 gglucose solution (50% w/w) and ampicillin 16 mL stock solution (25mg/mL).

The dissolved oxygen (dO) concentration was controlled at 25% of airsaturation. The dO was controlled first by impeller agitation rate (400to 1400 rpm) and later by aeration rate (2 to 10 slpm). The pH wascontrolled at 6.8. NH₄OH (29% w/w) and H₂SO₄ (20% w/v) were used for pHcontrol. The temperature was controlled at 35° C. and the head pressurewas 0.5 bars. At ca 6 OD₅₅₀ the culture was transferred to the 200 LBiostat-D Braun fermenter. The medium used was the same as in the seedfermenter, the initial working volume was 140 L and 50% w/w glucose wascharged to 8 g/L. The fermentation started as a batch operation, andonce the glucose was depleted (<0.5 g/L) a fed batch operation with 50%w/w glucose was initiated with a predetermined rate (Table 6), at ca 25OD₅₅₀ the feed was switched to 25% D-lactose solution with apre-determined rate (Table 7).

The temperature was controlled at 35.0° C., the head pressure at 0.5bar, the pH at 1^(st) stage (glucose phase) at 6.8 and at the 2^(nd)stage (lactose phase) at 7.2, NH₄OH (29% w/w) and H₂SO₄ (20% w/v) wereused for pH control, the dO controlled at 1^(st) stage at 25% of airsaturation and 2^(nd) stage at 10%, the dO was controlled by agitationfirst (250-450 rpm) and later by aeration (25-35 slpm). Glucose andlactose levels were monitored during the fed operation and if the levelsof glucose exceeds 0.1 g/L or lactose above 1 g/L the feed program waseither temporarily halted or reduced. The run was ended 40 h after theinitiation of lactose feed, and cells were either harvested bycentrifugation or microfiltration or kept in the vessel for treatmentwith glutaraldehyde. The fermentation produced about 8 kg dry cellweight with a nitrilase specific activity of 2819 BZN U/g dcw (1788 GLNU/g dcw).

TABLE 7 Feed protocol Feed time Feed intervals rate (h) g/min SubstrateStage 0 6.13 50% w/w glucose 1^(st) 1 7.13 50% w/w glucose 1^(st) 2 8.2850% w/w glucose 1^(st) 3 9.62 50% w/w glucose 1^(st) 4 11.18 50% w/wglucose 1^(st) 5 11.18 50% w/w glucose 1^(st) 6 11.18 50% w/w glucose1^(st) 7 11.18 50% w/w glucose 1^(st) 8 11.18 50% w/w glucose 1^(st) 011.22 25% w/w lactose 2^(nd) 2 24.42 25% w/w lactose 2^(nd) 20 16.72 25%w/w lactose 2^(nd) 30 18.7 25% w/w lactose 2^(nd) 40 18.7 25% w/wlactose 2^(nd)

At the end of the fermentation, the agitation was reduced to 150 rpm,the aeration stopped and the temperature maintained at 35° C. Part ofthe fermentation broth was withdrawn, leaving ca. 180 kg in thefermenter. This remaining broth was titrated to pH 5.2 and maintained atthis pH with 20% H₂SO₄ (20% w/w) and NaOH (50% w/w) while 9.0 L ofaqueous glutaraldehyde (GA, 10% w/w) was added with stirring at a rateof ˜90 mL/min; this rate of addition was equivalent to 50 mgglutaraldehyde/L fermentation broth/min, and the final concentration ofglutaraldehyde was ca. 5 g glutaraldehyde/L (0.035 gglutaraldehyde/OD₅₅₀). After 5 h from initiation of glutaraldehydeaddition to the broth, the pH was adjusted to 7.0, and 1.8 L of aqueoussodium bisulfite (10% w/w, pH 7) was added (ca. 1 g sodium bisulfite/Lfinal concentration) with stirring, and the broth stirred for anadditional 15 min. The temperature of the broth was then decreased to10° C., and the agitation decreased to 100 rpm. The broth wasconcentrated to 40 kg of cell suspension using a Diskstack centrifuge(Alfa Laval), then 50 kg Di water (20° C.) was added to the suspensionand the mixture was concentrated by centrifugation to produce 40 kg ofwashed cell suspension. The suspension (identified as NIT 188A-C2) wasstored at 5° C., and a portion of the cell suspension was used directlyfor the preparation of an immobilized cell catalyst (Example 6).Nitrilase specific activity during each process step is summarized inTable 8.

TABLE 8 Nitrilase activity during different stages of GA and bisulfitetreatment fermentation stage BZN U/g dcw pre GA treatment 2819 post GA3300 post NaHSO3 2493

EXAMPLE 4 Immobilization of Glutaraldehyde Pretreated E. coliMG1655/INM18-168V in GA/PEI-cross-linked Carrageenan Beads

The final cell suspension concentrate recovered from the glutaraldehydeand sodium bisulfite-treated fermentation broth of Example 5 wascentrifuged at 5° C. The resulting cell pellet was re-suspended in a5-fold by weight amount of 0.35 M potassium phosphate buffer (pH 7.2),and centrifugation of the resulting cell suspension at 5° C. produced awet cell paste that was immobilized and chemically cross-linked with GAand PEI as described in Example 2. The resulting immobilized cellcatalyst was identified as immobilized NIT 188A-C2.

EXAMPLE 5 Improvement in Biocatalyst Specific Activity in ConsecutiveBatch Reactions with Catalyst Recycle usingGlutaraldehyde/polyethylenimine Cross-Linked Carrageenan-immobilized E.coli MG1655/ISW138-F168V Transformant

In a typical procedure, duplicate sets of batch reactions for theconversion of glycolonitrile to glycolic acid were run in 50-mL jacketedreaction vessels equipped with overhead stirring and temperaturecontrol. Each reactor was charged with 8 g of GA/PEI-cross-linked E.coli MG1655/pSW138-168V/carrageenan beads (prepared using the process asdescribed in Example 1 (no GA pretreatment prior to immobilization) orExample 4 (GA pretreatment prior to immobilization)) containing 5% (dcw)transformant expressing the A. facilis 72W nitrilase mutant F168V (SEQID NO: 51). To the vessel was then added 14.78 mL of distilled water and6.0 mL of aqueous ammonium glycolate (4.0 M, pH 7.0), and the reactionvessel flushed with nitrogen. The mixture was stirred at 25° C. whileprogrammable syringe pumps were used to simultaneously add 1.07 mL of 60wt % glycolonitrile (GLN) in water (12.0 mmol GLN, 0.084 mmolformaldehyde; Fluka (redistilled, stabilized with 0.5 wt % glycolicacid)) and 0.150 mL of aqueous ammonium hydroxide (1.875 wt %); oneequivalent volume of GLN and ammonium hydroxide solutions was addedsimultaneously every 2 h (for a total of eight equivalent additions eachof GLN solution and aqueous ammonium hydroxide) to maintain theconcentration of GLN at ≦400 mM and the pH within a range of 6.5-7.5.Four 0.050-mL reaction samples were removed at pre-determined timesafter the first GLN addition and analyzed by HPLC to determine theinitial reaction rate and the catalyst specific activity (μmol glycolicacid/min/g dcw biocatalyst). At completion of the reaction, there was100% conversion of GLN to produce glycolic acid (as the ammonium salt)in >99% yield.

At the end of the first reaction, the aqueous product mixture wasdecanted from the catalyst (under nitrogen), leaving ca. 10.3 g of amixture of immobilized cell catalyst (8.0 g) and remaining productmixture (ca. 2.3 g). To the reaction vessel then added 20.78 mL ofdistilled, deionized water, and a second reaction was performed at 25°C. by the addition of aliquots of aqueous GLN and ammonium hydroxide asdescribed immediately above. The specific activities of recoveredbiocatalyst in consecutive batch reactions with catalyst recycle arelisted in Table 7.

TABLE 7 Dependence of recovered biocatalyst specific activity inconsecutive batch reactions with catalyst recycle on glutaraldehydepretreatment of cells prior to immobilization. decrease in specificimmobilized glutaraldehyde biocatalyst specific activity (GLN U/g dcw)activity, cell pretreatment in consecutive batch reactions rxn1 tobiocatalyst of cells reaction 1 reaction 2 reaction 3 reaction 4 rxn4(%) NIT 188C2 yes 1826 1518 1596 1759 4 NIT 188C2 yes 1857 1656 15811947 0 NIT 027 no 1312 872 898 816 38 NIT 027 no 1416 856 931 621 56 NIT49 no 1694 702 718 886 48 NIT 49 no 1665 868 869 946 43 NIT 60-A no 1319730 744 563 57 NIT 60-A no 1410 750 725 699 50 NIT 60-B no 1222 757 857802 34 NIT 60-B no 1335 722 839 808 39

EXAMPLE 6 Storage Stability of Glutaraldehyde/polyethylenimineCross-linked Carrageenan-immobilized E. coli MG1655/ISW138-F168VPrepared Using Glutaraldehyde Pretreated Cells

Freshly-prepared glutaraldehyde/polyethylenimine cross-linkedcarrageenan-immobilized E. coli MG1655/pSW138-F168V prepared usingglutaraldehyde pretreated cells (as described in Example 4) were storedfor 32 days in 1.0 M ammonium bicarbonate (pH 7.3) at 5° C. Prior touse, the beads were washed twice for 15 min with 180 mL of 0.1 Mammonium glycolate (pH 7.0) at room temperature. Biocatalyst stored foreither 4 days or 32 days at 5° C. were each evaluated in duplicate setsof consecutive batch reactions with biocatalyst recycle using theprocedure described in Example 5 (Table 8).

TABLE 8 Specific activity of biocatalyst prepared using glutaraldehydepretreatment of cells prior to immobilization in consecutive batchreactions with catalyst recycle, using biocatalyst stored at 5° C. for 4or 32 days. decrease in specific immobilized biocatalyst specificactivity (GLN U/g dcw) activity, cell days stored at in consecutivebatch reactions rxn1 to biocatalyst 5° C. reaction 1 reaction 2 reaction3 reaction 4 rxn4 (%) NIT 188C2 4 1826 1518 1596 1759 4 NIT 188C2 4 18571656 1581 1947 0 NIT 188C2 32 1910 1455 1580 1543 19 NIT 188C2 32 19871434 1472 1783 10

1. A process for improving the retention of the initial specificactivity of an enzyme catalyst having nitrilase activity during theconversion of glycolonitrile to glycolic acid, said process comprising:(a) producing an enzyme catalyst having nitrilase activity byfermentation, said enzyme catalyst comprising a polypeptide having anamino acid sequence selected from the group consisting of SEQ ID NOs: 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, and57; (b) pretreating said enzyme catalyst with glutaraldehyde; (c)optionally inactivating unreacted glutaraldehyde with bisulfitefollowing glutaraldehyde pretreatment; (d) recovering the enzymecatalyst from (b) or (c) and immobilizing said enzyme catalyst incarrageenan; (e) cross-linking the resulting carrageenan-immobilizedenzyme catalyst of (d) with glutaraldehyde and polyethylenimine; and (f)producing a glutaraldehyde-pretreated immobilized and cross-linkedenzyme catalyst, wherein said glutaraldehyde-pretreated immobilized andcross-linked enzyme catalyst has improved retention of initial specificactivity during conversion of glycolonitrile to glycolic acid ascompared to non-glutaraldehyde-pretreated immobilized and cross-linkedenzyme catalysts under the same reaction conditions.
 2. The process ofclaim 1 wherein the pH is maintained between 5.0 and 9.0 duringpretreatment with glutaraldehyde.
 3. The process of claim 1 wherein thepretreating with glutaraldehyde in step (b) comprises addingglutaraldehyde to a fermentation broth produced by step (a) in an amountin the range of about 3 g/L (0.025 g GA per OD₅₅₀) and about 5 g/L(0.042 g GA per OD₅₅₀).
 4. The process of claim 1 wherein thepretreating with glutaraldehyde in step (b) comprises addingglutaraldehyde to a fermentation broth produced by step (a) at a rate of50 mg/L/h to 500 mg/L/h.
 5. An isolated glutaraldehyde pretreated,immobilized and cross-linked enzyme catalyst produced by the process ofclaim
 1. 6. The enzyme catalyst of claim 5, wherein said catalystretains at least about 70% of its initial specific activity after atleast four consecutive batch recycle reactions.